D biotin

We subcloned the VHH gene fragment to a pBAD vector after digestion by NcoI and BstEII restriction endonucleases and co-transformed the ligated material into WK6 cells with pBirA plasmid [19 (link)]. The expression of VHH-BAD fusion protein was induced with 1 mM IPTG followed by the addition of 50 μM d-biotin (Bio Basic Inc., Beijing, China) and incubated for 30 min. Cells were collected and the periplasmic proteins extracted by osmotic shock. Soluble nanobodies, which were coupled to biotin, were purified by Streptavidin Mutein Matrix (Roche, Basel, Germany). After washing with PBS, the nanobodies conjugated with biotin were eluted by 6 mM d-biotin and dialyzed into PBS.

The influenza H5N1 virus used in this study was obtained from the Shanghai Institute of Biological Products (China). Freund’s complete adjuvant and Freund’s incomplete adjuvant were purchased from Sigma-Aldrich (USA). Fast Track 2.0 Kit was provided by Invitrogen (USA) and Oligo_dT primer was obtained from Thermo Scientific (USA). Restriction enzymes Pst I, Not I, Nco I and BstE II were provided from NEB (USA). The mouse anti-HA tag antibody was purchased from Covance (New Zealand). The anti-mouse IgG-alkaline phosphatase, bis phosphate and His-select column were purchased from Sigma-aldrich (USA). The D-biotin and horseradish peroxidase (HRP) were purchased from Bio Basic Inc. (China). The Streptavidin Mutein Matrix was purchased from Roche (Switzerland). The microtiter plate was purchased from Thermo Scientific NUNC (Denmark). The BeaverNano™ Streptavidin Matrix Coated 96-Well plates were provided by BeaverNano technology company (China). The VCSM13 helper phages, TG1 cells, WK6 cells, plasmid pBAD and plasmid pBirA were obtained from Prof. Serge Muyldermans’s lab (Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Belgium).